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In Photosystem II electrons from water splitting pass through a primary quinone electron acceptor (QA) to the secondary plastoquinone (QB). The D2 protein forms the QA-binding site and the D1 protein forms the QB-binding site. A non-heme iron sits between QA and QB resulting in a quinone-Fe-acceptor complex that must be activated before assembly of the oxygen-evolving complex can occur. An extended loop (residues 223-266) between the fourth (helix D) and fifth (helix E) helices of the D1 protein activates forward electron transfer via a conformational change that stabilizes a bidentate bicarbonate ligand to the non-heme iron while simultaneously stabilizing the binding of QB. We show that positioning of D1:Phe265 to provide a hydrogen bond to the distal oxygen of QB is required for forward electron transfer. In addition, mutations targeting D1:Phe265, resulted in a 50 mV decrease in the QB/QB- midpoint potential.

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Plants absorb light energy for photosynthesis via photosystem complexes in their chloroplasts. However, excess light can damage the photosystems and decrease photosynthetic output, thereby inhibiting plant growth and development. Plants have developed a series of light acclimation strategies that allow them to withstand high light. In the first line of defense against excess light, leaves and chloroplasts move away from the light and the plant accumulates compounds that filter and reflect the light. In the second line of defense, known as photoprotection, plants dissipate excess light energy through non-photochemical quenching, cyclic electron transport, photorespiration, and scavenging of excess reactive oxygen species. In the third line of defense, which occurs after photodamage, plants initiate a cycle of photosystem (mainly photosystem II) repair. In addition to being the site of photosynthesis, chloroplasts sense stress, especially light stress, and transduce the stress signal to the nucleus, where it modulates the expression of genes involved in the stress response. In this review, we discuss current progress in our understanding of the strategies and mechanisms employed by plants to withstand high light at the whole-plant, cellular, physiological, and molecular levels across the three lines of defense.

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Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2's ß-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4.

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Recent studies have indicated a good potential for using solar-induced chlorophyll fluorescence (SIF) to estimate photosynthetic CO2 assimilation. SIF can be emitted by both Photosystem I (PSI) and Photosystem II (PSII), but it is the SIF signals from PSII which are related to photosynthetic carbon fixation. However, since top-of-canopy SIF observations (SIFTOC) always contain contributions from both photosystems, to mechanistically estimate gross primary productivity (GPP) from SIF, it is essential to extract PSII SIF from SIFTOC. Based on the differences in the relative contribution of PSII across different wavelengths, we propose a practical approach for extracting PSII contribution to SIFTOC at the near-infrared (NIR) band (fPSII_760) using measurements of SIFTOC in the red and NIR spectral regions. A leaf-scale concurrent instrument was developed to assess the response of fPSII_760 under varying environments. For winter-wheat leaves, as light intensity increased from 0 to 400 µmol m-2 s-1, fPSII_760 rose from 0.6 to 0.8; with further increase in light intensity to 1800 µmol m-2 s-1, fPSII_760 consistently decreased to 0.65. There was a slight decreasing trend in fPSII_760 with rising temperatures, with values dropping from 0.65 at 15 °C to 0.61 at 40 °C. We found that variations in fPSII_760 are due to changes in the fluorescence yield of PSII, with the two having a positively proportional relationship. We also estimated canopy-scale fPSII_760 for a winter-wheat study site: fPSII_760 varied from 0.61 to 0.83, with a mean value of 0.78 during the peak growing season. A comparison with eddy covariance-derived GPP reveals that GPP estimated with dynamic fPSII_760 was more accurate than that calculated using fixed fPSII_760, with R2 increasing from 0.6 to 0.84. This study contributes to a deeper understanding of the link between SIF and photosynthetic CO2 assimilation, paving the way for more effective use of SIF to estimate GPP.

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The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H2). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O2 sensitivity, binding specificity, or H2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS]4 linker group (CbHydA1-PsaE). We demonstrate that the CbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H2 at a rate of 84.9 ± 3.1 µmol H2 mgchl-1 h-1. Further, when prepared and illuminated in the presence of O2, the nanoconstruct retains the ability to generate H2, though at a diminished rate of 2.2 ± 0.5 µmol H2 mgchl-1 h-1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O2-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H2 generating system that can function in the presence of O2.

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Hexavalent chromium (Cr (VI)) is a heavy metal that is distributed globally and poses a significant threat to the environment through various mechanisms. It can react with soil and water, leading to severe environmental damage. In this study, the toxicity of Cr (VI) was investigated by analyzing two major cyanobacteria species, Nostoc commune and Anabaena variabilis, commonly found in soil along with their consortia. The findings revealed that the toxicity mechanisms of Cr (VI) differed in individual monocultures, with Cr (VI) competing with various components. However, when the cyanobacteria species were combined, i.e., in consortia, they demonstrated an impressive retention of their functioning even in Cr (VI) concentration at 10 ppm. The study also concluded that non-photochemical quenching played a critical role in minimizing Cr (VI) toxicity. Furthermore, the research examined the role of the S-cycle in the process. The quantum yield of electron flux revealed that the Cr (VI) was competing with Qa in A. variabilis and with Qb in N. commune, albeit the photosystem dysfunction is only visible in the latter. The mechanism seemed to be quantum tunneling alteration because of the Cr (VI) having different energized quantum wells. The consortia proved to be behaving in a better manner as compared to the control. Overall, this study reveals the mode of toxicity of Cr (VI) in these two important cyanobacterial strains as well as it also discusses the mechanism of tolerance of consortia against Cr (VI) toxicity.

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The daily light-dark cycle is a recurrent and predictable environmental phenomenon to which many organisms, including cyanobacteria, have evolved to adapt. Understanding how cyanobacteria alter their metabolic attributes in response to subjective light or dark growth may provide key features for developing strains with improved photosynthetic efficiency and applications in enhanced carbon sequestration and renewable energy. Here, we undertook a label-free proteomic approach to investigate the effect of extended light (LL) or extended dark (DD) conditions on the unicellular cyanobacterium Crocosphaera subtropica ATCC 51142. We quantified 2287 proteins, of which 603 proteins were significantly different between the two growth conditions. These proteins represent several biological processes, including photosynthetic electron transport, carbon fixation, stress responses, translation, and protein degradation. One significant observation is the regulation of over two dozen proteases, including ATP dependent Clp-proteases (endopeptidases) and metalloproteases, the majority of which were upregulated in LL compared to DD. This suggests that proteases play a crucial role in the regulation and maintenance of photosynthesis, especially the PSI and PSII components. The higher protease activity in LL indicates a need for more frequent degradation and repair of certain photosynthetic components, highlighting the dynamic nature of protein turnover and quality control mechanisms in response to prolonged light exposure. The results enhance our understanding of how Crocosphaera subtropica ATCC51142 adjusts its molecular machinery in response to extended light or dark growth conditions.

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Photosystem I (PSI) serves as a model system for studying fundamental processes such as electron transfer (ET) and energy conversion, which are not only central to photosynthesis but also have broader implications for bioenergy production and biomimetic device design. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate key light-induced charge separation steps in PSI isolated from several green algal and cyanobacterial species. Following photoexcitation, rapid sequential ET occurs through either of two quasi-symmetric branches of donor/acceptor cofactors embedded within the protein core, termed the A and B branches. Using high-frequency (130 GHz) time-resolved EPR (TR-EPR) and deuteration techniques to enhance spectral resolution, we observed that at low temperatures prokaryotic PSI exhibits reversible ET in the A branch and irreversible ET in the B branch, while PSI from eukaryotic counterparts displays either reversible ET in both branches or exclusively in the B branch. Furthermore, we observed a notable correlation between low-temperature charge separation to the terminal [4Fe-4S] clusters of PSI, termed FA and FB, as reflected in the measured FA/FB ratio. These findings enhance our understanding of the mechanistic diversity of PSI's ET across different species and underscore the importance of experimental design in resolving these differences. Though further research is necessary to elucidate the underlying mechanisms and the evolutionary significance of these variations in PSI charge separation, this study sets the stage for future investigations into the complex interplay between protein structure, ET pathways, and the environmental adaptations of photosynthetic organisms.

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The process of oxygenic photosynthesis is primarily driven by two multiprotein complexes known as photosystem II (PSII) and photosystem I (PSI). PSII facilitates the light-induced reactions of water-splitting and plastoquinone reduction, while PSI functions as the light-driven plastocyanin-ferredoxin oxidoreductase. In contrast to the highly conserved structure of PSII among all oxygen-evolving photosynthetic organisms, the structures of PSI exhibit remarkable variations, especially for photosynthetic organisms that grow in special environments. In this review, we make a concise overview of the recent investigations of PSI from photosynthetic microorganisms including prokaryotic cyanobacteria and eukaryotic algae from the perspective of structural biology. All known PSI complexes contain a highly conserved heterodimeric core; however, their pigment compositions and peripheral light-harvesting proteins are substantially flexible. This structural plasticity of PSI reveals the dynamic adaptation to environmental changes for photosynthetic organisms.

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The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants' capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production.

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This review comprehensively examines the phenomenon of photoinhibition in plants, focusing mainly on the intricate relationship between photodamage and photosystem II (PSII) repair and the role of PSII extrinsic proteins and protein phosphorylation in these processes. In natural environments, photoinhibition occurs together with a suite of concurrent stress factors, including extreme temperatures, drought and salinization. Photoinhibition, primarily caused by high irradiance, results in a critical imbalance between the rate of PSII photodamage and its repair. Central to this process is the generation of reactive oxygen species (ROS), which not only impair the photosynthetic apparatus first PSII but also play a signalling role in chloroplasts and other cellulular structures. ROS generated under stress conditions inhibit the repair of photodamaged PSII by suppressing D1 protein synthesis and affecting PSII protein phosphorylation. Furthermore, this review considers how environmental stressors exacerbate PSII damage by interfering with PSII repair primarily by reducing de novo protein synthesis. In addition to causing direct damage, these stressors also contribute to ROS production by restricting CO2 fixation, which also reduces the intensity of protein synthesis. This knowledge has significant implications for agricultural practices and crop improvement under stressful conditions.

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Manganese (Mn) is considered as an essential element for plant growth. Mn starvation has been shown to affect photosystem II, the site of the Mn4CaO5 cluster responsible for water oxidation. Less is known on the effect of Mn starvation on photosystem I. Here we studied the effects of Mn deficiency in vivo on redox changes of P700 and plastocyanin (Pc) in the liverwort Marchantia polymorpha using the KLAS-NIR spectrophotometer. Far-red illumination is used to excite preferentially photosystem I, thus facilitating cyclic electron transport. Under Mn starvation, we observed slower oxidation of P700 and a decrease in the Pc signal relative to P700. The lower Pc content under Mn deficiency was confirmed by western blots. Re-reduction kinetics of P700+ and Pc+ were faster in Mn deficient thalli than in the control. The above findings show that the kinetics studied under Mn deficiency not only depend on the number of available reductants but also on how quickly electrons are transferred from stromal donors via the intersystem chain to Pc+ and P700+. We suggest that under Mn deficiency a structural reorganization of the thylakoid membrane takes place favoring the formation of supercomplexes between ferredoxin, cytochrome b6f complex, Pc and photosystem I, and thus an enhanced cyclic electron transport.

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Intertidal macroalgae are sessile poikilohydric organisms exposed to desiccation stress during emersion. Water relations parameters are useful tools to evaluate an organism's capacity to withstand water scarcity conditions, but such information on marine intertidal macroalgae is scarce. We assessed the water relations of the intertidal relict Fucus virsoides, the unique Fucus species endemic to the Mediterranean. We combined measurements of water potential (Ψ) parameters derived from pressure-volume curves and chlorophyll a fluorescence (Fv/Fm) in juvenile and adult thalli sampled in three different dates between March and April 2023. F. virsoides exhibited remarkable water stress tolerance, as evidenced by the low water potential at turgor loss point (Ψtlp, -7.0 MPa on average), and the maintenance of high Fv/Fm at low water potentials indicating a prolonged maintenance of healthy physiological status. While no differences were observed between growth stages, Ψtlp, capacitance (C) and the bulk modulus of elasticity (ε) varied significantly according to the sampling dates, whereas the osmotic potential at full turgor did not significantly change. Ψ measured on thalli collected after a typical prolonged emersion period was markedly lower (-12.3 MPa on average) than the estimated Ψtlp, suggesting that the population is frequently undergoing turgor loss. Further investigations are required to determine environmental tolerance ranges based on water status characteristics to enhance our understanding of F. virsoides responses and vulnerability to climate change, thus providing insight into the possible causes of its widespread decline.

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Adaptation and functional significance of chlorophyll deficit in the light green leaf sectors of variegated plants are little known. Efficiency of photosystem II for dark and light adapted states (Fv/Fm and ΔF/Fm') and fluorescence decrease rates (Rfd) of light green leaf sectors of Dracaena fragrans L. were studied by methods of PAM-fluorometry and video registration. In addition, white light reflectance and transmittance of these leaf sectors were measured using an integrating sphere. Absorption was calculated from reflectance and transmittance. Net CO2 assimilation rates (PN) were measured using a flow chamber and photolytic O2 evolution rates (PAYO2) were studied by a novel method of Fourier photoacoustics which is insensitive to respiration, photorespiration and other processes of O2 uptake. All the photosynthetic parameters (Fv/Fm, ΔF/Fm', PN and PAYO2) were found to be very close between light green and normal green leaf sectors, whereas chlorophyll content and light absorption were 7.5-fold and 1.47-fold different respectively. Contradiction between low chlorophyll absorption and high (as in normal green sectors) rate of oxygenic photosynthesis in light-green sectors was proposed to be a consequence of different contribution of cyclic electron transport around PSII (CET-PSII) and/or around PSI (CET-PSI) in the total photosynthesis occurring in these sectors. Particularly, it cannot be excluded, that some part of CET activity occurring in normal green leaf sectors may be lost in the light green sectors retaining the same linear (non-cyclic) electron transport (LET) activity as in normal green sectors.

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Cold stress has severe negative consequences for plant growth and crop yield. Here, we report that an Arabidopsis thaliana mutant that lacks the HPE1 gene, which encodes an RNA-binding protein, maintains higher photosynthetic activity under cold stress, together with higher accumulation of thylakoid proteins. We showed that HPE1 interacts with MORF2 and MORF9 and thereby mediates RNA editing in chloroplasts. Loss of HPE1 function increased the editing efficiency at four RNA editing sites, rpoC-488, ndhB-149, ndhB-746 and matK-706, under cold stress and altered the expression of nuclear photosynthesis-related genes and cold-responsive genes. We propose that HPE1-mediated RNA editing acts as a trigger for retrograde signaling that affects photosynthesis under cold stress.

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The structure and mechanism of the water-oxidation chemistry that occurs in photosystem II have been subjects of great interest. The advent of X-ray free electron lasers allowed the determination of structures of the stable intermediate states and of steps in the transitions between these intermediate states, bringing a new perspective to this field. The room-temperature structures collected as the photosynthetic water oxidation reaction proceeds in real time have provided important novel insights into the structural changes and the mechanism of the water oxidation reaction. The time-resolved measurements have also given us a view of how this reaction-which involves multielectron, multiproton processes-is facilitated by the interaction of the ligands and the protein residues in the oxygen-evolving complex. These structures have also provided a picture of the dynamics occurring in the channels within photosystem II that are involved in the transport of the substrate water to the catalytic center and protons to the bulk.

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As a critical step in advancing the simulation of photosynthetic complexes, we present the Martini 3 coarse-grained (CG) models of key cofactors associated with light harvesting (LHCII) proteins and the photosystem II (PSII) core complex. Our work focuses on the parametrization of beta-carotene, plastoquinone/quinol, violaxanthin, lutein, neoxanthin, chlorophyll A, chlorophyll B, and heme. We derived the CG parameters to match the all-atom reference simulations, while structural and thermodynamic properties of the cofactors were compared to experimental values when available. To further assess the reliability of the parameterization, we tested the behavior of these cofactors within their physiological environments, specifically in a lipid bilayer and bound to photosynthetic complexes. The results demonstrate that our CG models maintain the essential features required for realistic simulations. This work lays the groundwork for detailed simulations of the PSII-LHCII super-complex, providing a robust parameter set for future studies.

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Cyanobacteria move by gliding motility on surfaces toward the light or away from it. It is as yet unclear how the light direction is sensed on the molecular level. Diverse photoreceptor knockout mutants have a stronger response toward the light than the wild type. Either the light direction is sensed by multiple photoreceptors or by photosystems. In a study on photophobotaxis of the filamentous cyanobacterium Phormidium lacuna, broad spectral sensitivity, inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and a highly sensitive response speaks for photosystems as light direction sensors. Here, it is discussed whether the photosystem theory could hold for phototaxis of other cyanobacteria.

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Photosystem I (PSI) is an essential protein complex for oxygenic photosynthesis and is also known to be an important source of reactive oxygen species (ROS) in the light. When ROS are generated within PSI, the photosystem can be damaged. The so-called PSI photoinhibition is a lethal event for oxygenic phototrophs, and it is prevented by keeping the reaction center chlorophyll (P700) oxidized in excess light conditions. Whereas regulatory mechanisms for controlling P700 oxidation have been discovered already, the molecular mechanism of PSI photoinhibition is still unclear. Here, we characterized the damage mechanism of PSI photoinhibition by in vitro transient absorption and electron paramagnetic resonance (EPR) spectroscopy in isolated PSI from cucumber leaves that had been subjected to photoinhibition treatment. Photodamage to PSI was induced by two different light treatments: 1. continuous illumination with high light at low (chilling) temperature (C/LT) and 2. repetitive flashes at room temperature (F/RT). These samples were compared to samples that had been illuminated with high light at room temperature (C/RT). The [FeS] clusters FX and (FA FB) were destructed in C/LT but not in F/RT. Transient absorption spectroscopy indicated that half of the charge separation was impaired in F/RT, however, low-temperature EPR revealed the light-induced FX signal at a similar size as in the case of C/RT. This indicates that the two branches of electron transfer in PSI were affected differently. Electron transfer at the A-branch was inhibited in F/RT and also partially in C/LT, while the B-branch remained active.

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